This invention relates to a process for producing .beta.-stevioside derivatives. More specifically, this invention relates to a process for producing .beta.-1,3 or .beta.-1,4-glycosyl stevioside which comprises reacting stevioside with a .beta.-1,3- or .beta.-1,4-glycosyl sugar compound in aqueous solution or suspension (to be referred to inclusively as aqueous solution) in the presence of .beta.-1,3- or .beta.-1,4-glycosyltransferase or a microorganism having .beta.-1,3- or .beta.-1,4-glycosyl transferring activity. According to an especially preferred embodiment, this invention pertains to a process for producing stevioside derivatives intended for the production of rebaudioside A by a fermentation technique.
In recent years, the use of such artificial sweeteners as sodium cyclamate, sodium saccharin and dulcin, in food has been prohibited or restricted from the standpoint of safety, and on the other hand, an adverse effect of the excessive intake of sugar on health has become a problem. It has been eagerly desired therefore to develop natural sweeteners which would replace these artificial ones. Under the circumstances, a demand for stevioside has rapidly increased because unlike sugar, it is a sweetener of a low caloric value and has a sweetness ratio about 300 times as high as that of sugar. Stevioside is a sweetening component (contained in an amount of 6 to 12%) extracted from Stevia rebaudiana BERTONI (to be referred to simply as Stevia) belonging to the family Compositae, and is a .beta.-glucosyl glycoside in which the aglycon is steviol. Stevioside is now used to sweeten foods and drinks. The sweetness of stevioside appears more slowly than sugar, and remains longer as an unpleasant aftertaste. Furthermore, it also has bitterness and astringency. It is thought that because of such a defect, there is a limit to its usage and its amount used, and some improvement in the quality of its sweetness is required. Attempts are made to improve the quality of the sweetness of stevioside by adding at least one natural sweetener such as sucrose, glucose or fructose, or by adding an amino acid or its hydrochloride. According to these methods, the amounts of the aforesaid additives should be made extremely large in order to reduce the bitterness and astringency of the extract of Stevia. Subsequently, the characteristics of stevioside as a low-calorie sweetener are lost.
Rebaudioside A (.beta.-1,3-monoglucosyl stevioside) is contained in an amount of about 2 to 6% in the leaves or stalks of Stevia, and is the second highest in content following stevioside (6 to 12%), a main ingredient. Heretofore, a mixture of these ingredients has been used as a Stevia sweetener. Stevioside has bitterness and leaves an unpleasant aftertaste, whereas rebaudioside A has mild sweetness without bitterness and has a higher sweetness ratio to sucrose than stevioside.
Recently, a Stevia sweetener having improved taste and being free from bitterness without an increase in calorie was developed by forming an .alpha.-glycosyl stevioside (U.S. Pat. No. 4,219,571). This sweetener, however, has the defect that its sweetness ratio is lower than stevioside. Since this sweetener has added thereto an .alpha.-glucosyl sugar compound unlike a .beta.-glycosyl sugar compound in the present invention, it has "sticky" sweetness different from the sweetness of sugar. In contrast, the sweetener in accordance with this invention having a .beta.-glycosyl sugar compound added thereto has sharp sweetness which is close to that of sugar.
Heretofore, rebaudioside A has been obtained by a method which comprises obtaining a mixture of stevioside and rebaudioside A by extraction from the dried leaves of Stevia followed by purification, removing stevioside from the mixture by crystallization, and repeatedly re-crystallizing the residue. Since the yield of the rebaudioside A is poor, it is necessary to increase the content of rebaudioside A when extracting the aforesaid mixture. An attempt has been made to obtain Stevia leaves having an increased content of rebaudioside A by breeding Stevia, but because of the instability of maintaining the character by heredity, this technique has little practical utility at present.
The present inventors, therefore, have made extensive investigations in order to convert stevioside present in the largest amount in the extract of Stevia leaves into .beta.-glucosyl stevioside and .beta.-galactosyl stevioside by the addition-reaction of glucose or galactose with it by a fermentation or enzyme technique.